Nitric oxide-releasing polymers

ABSTRACT

This invention relates to compositions comprising carbon-based diazeniumdiolates attached to hydrophobic polymers that releases nitric oxide (NO). The carbon-based diazeniumdiolated polymers release NO spontaneously under physiological conditions without subsequence nitrosamine formation. The present invention also relates to methods of preparing the carbon-based diazeniumdiolated polymers, compositions comprising such polymers, methods of using such compositions, and devices employing such polymer compositions.

This application claims priority under 35 U.S.C. §120 to U.S.Provisional Application No. 60/542,277 filed Feb. 9, 2004, and to U.S.Provisional Application No. 60/542,298 filed Feb. 9, 2004, each of whichis incorporated herein by reference in its entirety.

This work was sponsored by U.S. Public Health Service Grant No. R44HL062729 from the National Heart Lung and Blood Institute of TheNational Institutes of Health.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to nitric oxide-releasingpolymers. More specifically, the present invention relates tocarbon-based diazeniumdiolate nitric oxide-releasing polymers. Thepresent invention also provides methods for a novel class of coatings inwhich NO-releasing carbon-based diazeniumdiolates may be covalentlylinked to a surface, whereby the release of NO imparts increasedbiocompatibility or other beneficial properties to the coated surface.One possible preferred application for this class of coatings would bein medical devices.

Nitric oxide (NO) is a bioregulatory molecule with diverse functionalroles in cardiovascular homeostasis, neurotransmission and immuneresponse (Moncada et al., 1990; Marletta et al., 1990). Because NOinfluences such a vast array of physiological activity, it is desirableto have compounds release NO for use as drugs and physiological andpharmacological research tools. Even more desirable are non-toxic,non-carcinogenic compounds that can generate NO under physiologicalconditions for therapeutic and clinical applications. Such compounds,however, have been difficult to develop.

Small molecules (generally described as molecules with Formula Weightsless than 600) that release NO are well known, and some classes such asthe organic nitrates have been used for decades therapeutically. These,however, are difficult to administer as they may circulate throughoutthe body causing a myriad of physiological effects leading todisturbances of homeostasis. For many therapeutic applications a morelocalized release of NO would be preferred.

More recently, polymeric forms of NO-releasing compounds have beendescribed where the NO donor molecule is part of, associated with,incorporated in, or otherwise bound to a polymer matrix. The vastmajority of polymeric NO donors are of the nitrogen- or N-baseddiazeniumdiolate class disclosed in U.S. Pat. No. 5,405,919, Keefer andHrabie; U.S. Pat. No. 5,525,357, Keefer et al; U.S. Pat. No. 5,632,981,Saavedra et al.; U.S. Pat. No. 5,676,963 Keefer and Hrabie; U.S. Pat.No. 5,691,423, Smith et al.; U.S. Pat. No. 5,718,892 Keefer and Hrabie;U.S. Pat. No. 5,962,520, Smith and Rao; U.S. Pat. No. 6,200,558,Saavedra et al.; U.S. Pat. No. 6,270,779, Fitzhugh et al.; U.S. PatentApplication Publication; Pub. No.: US 2003/0012816 A1, West and Masters.Diazeniumdiolates are a class of compounds which contain the —[N(O)NO]—functional group and have been known for over 100 years (Traube, 1898).

While N-based diazeniumdiolate polymers have the advantages of localizedspontaneous and generally controllable release of NO under physiologicalconditions, a major disadvantage associated with all N-baseddiazeniumdiolates is their potential to form carcinogenic nitrosaminesupon decomposition as shown in Equation 1 (Parzuchowski et al., 2002).Some nitrosamines are extremely carcinogenic and the potential fornitrosamine formation limits the N-based diazeniumdiolate class of NOdonors from consideration as therapeutic agents based on safety issues.

Other non-diazeniumdiolate forms of polymeric NO donors have beendescribed including S-nitroso compounds (U.S. Pat. Nos. 5,770,645 and6,232,434, Stamler et al.) and C-nitroso compounds (U.S. Pat. No.5,665,077, Rosen et al.; and U.S. Pat. No. 6,359,182, Stamler et al.).Regarding the S-nitroso compounds, their therapeutic potential islimited due to their rapid and unpredictable decomposition (release ofNO) in the presence of trace levels of Cu(I) and possibly Cu(II) ions(Dicks et al., 1996; Al-Sa'doni et al., 1997). Furthermore, S-nitrosocompounds may decompose by direct transfer of NO to reduced tissuethiols (Meyer et al., 1994; Liu et al., 1998). Finally, many mammalianenzymes may catalyze the release of NO from S-nitroso compounds(Jourd″heuil et al, 1999a; Jourd″heuil et al., 1999b; Askew et al.,1995; Gordge et al., 1996; Freedman et al., 1995; Zai et al., 1999;Trujillo et al., 1998). However tissue and blood levels of ions,enzymes, and thiols are subject to a wide range of variability in eachindividual, making the release of NO unpredictable from subject tosubject. The dependence and sensitivity of NO release on blood andtissue components limits the therapeutic potential of nitroso compoundsin medicine.

Several references to carbon- or C-based diazeniumdiolate smallmolecules (small molecules are generally described as molecules with aFormula Weight of 600 or less) which release NO have been disclosed(U.S. Pat. Nos. 6,232,336; 6,511,991; 6,673,338; Arnold et al. 2000;Arnold et al. 2002a; Arnold et al. 2002b). C-based diazeniumdiolates aredesirable because in contrast to N-based diazeniumdiolates they arestructurally unable to form nitrosamines while maintaining their abilityspontaneously release NO under physiological conditions. Furthermore,there have been recently published reports on NO-releasing imidates,methanetrisdiazeniumdiolate, and a bisdiazeniumdiolate derived from1,4-benzoquinone dioxime which released 2 moles of NO per mole ofcompound. (Arnold et al. 2000; Arnold et al. 2002a; Arnold et al.2002b). While the NO-releasing properties of these small molecules arefavorable, small molecules are very difficult to localize in the bodyafter administration and tend to diffuse easily throughout the body,resulting in possible systemic side effects of NO. An additional problemspecific to imidate- and thioimidate-derived molecules is that theprotein binding properties of imidates may be undesirable inapplications involving contact with blood, plasma, cells, or tissuebecause the imidate may react to form a covalent bond with tissueprotein (see below).

Recently, carbon- or C-based diazeniumdiolate polymers have beendisclosed (U.S. Pat. No. 6,673,338, Arnold et al., 2004). C-baseddiazeniumdiolates are desirable because in contrast to N-baseddiazeniumdiolate they are structurally unable to form nitrosamines whilemaintaining their ability spontaneously release NO under physiologicalconditions. Arnold et al. disclose imidates and thioimidates of thefollowing general structure (1):

where R¹ is a polymer in one embodiment. They also disclose embodimentswhere the imidate functional group is used to bind the molecule topolymers or biopolymers (proteins), as the imidate functional group iscommonly used to bind and/or cross-link proteins (Sekhar et al., 1991;Ahmadi and Speakman, 1978). However the protein binding properties ofimidates would be undesirable in applications involving contact withblood, plasma, cells, or tissue because the imidate may react withprotein tissue.

Thus there continues to be a need for NO-releasing polymers that releaseNO spontaneously under physiological conditions and in predictable andtunable quantities, where the NO release is not affected by metals,thiols, enzymes, or other tissue factors that may result in variable NOrelease, and where the polymer cannot decompose to form nitrosamines anddoes not covalently bind proteins.

Therefore, it is an object of the present invention to provide acomposition that includes a C-based diazeniumdiolate covalently attachedto a polymeric backbone that can generate localized fluxes of NOspontaneously under physiological conditions. It is a further object ofthe present invention to provide NO-releasing polymers that generatepredictable and tunable NO release rates. It is a further object of thepresent invention to provide diazeniumdiolate polymers that do notdecompose into nitrosamines or covalently bind proteins.

In addition, it is an object of the present invention to provide amethod of synthesis for the polymer bound C-based diazeniumdiolates. Afurther object of the present invention is to provide methods of use forthe C-based diazeniumdiolate polymers in biology and medicine. Furtherobjects and advantages of the invention will become apparent from thefollowing descriptions.

BRIEF SUMMARY OF THE INVENTION

The present invention accomplishes the above-described objects byproviding a polymer composition that spontaneously releases NO underphysiological conditions, without the possibility to form nitrosamines.The present invention provides a composition for the generation of NOfrom a C-based diazeniumdiolate that is covalently attached to aphenyl-containing polymer. The present inventors have developed analternative means of introducing the —[N(O)NO]⁻ functional group into apolymeric backbone by attachment of the —[N(O)NO]⁻ group to the polymervia a carbon atom, with the general formula:R³—C(R¹)_(x)(N₂O₂R²)_(y)  FORMULA 1where y may be 1-3 and x may be 0-2 and the sum of x plus y equals 3, R¹is not an imidate or thioimidate. R¹ may be represented by, but notlimited to an electron withdrawing group such as, but not limited to, acyano group; an ether group, such as, but not limited to —OCH₃, —OC₂H₅,and —OSi(CH₃)₃; a tertiary amine; or a thioether, such as, but notlimited to, —SC₂H₅, and —SPh (substituted or unsubstituted). The R¹group may also be a amine, such as, but not limited to, —N(C₂H₅)₂. R² isa countercation or organic group and R³ is a phenyl group. The phenylgroup may be pendant from the polymer backbone (as shown in Formula 2)or part of the polymer backbone (as shown in Formula 3). In addition tothe aforementioned advantages of this technology over the prior art,manipulation of the R¹ group in Formula 1 can alter the release kineticsand the amount of NO released. Alterations of the R¹ group to alter thequantity and kinetics of NO-released are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the quantity of NO released from 5.5 mg of cyano-modifiedchloromethylated polystyrene diazeniumdiolate in pH 7.4 buffer over a 15minute time period. Over this time period, 0.49 μmoles of NO per mgresin was produced. The quantity of NO released is measured in parts perbillion (ppb), which is assessed and measured as described herein.

FIG. 2 shows the quantity of NO-release from ethoxy-modifiedchloromethylated polystyrene diazeniumdiolate. This polymer compositionwas packed in 4 mm dialysis membrane (MWCO 3500), placed in a reactorvessel and submerged in pH 7.4 buffer. After 26 minutes the dialysistube was removed to demonstrate the absence of NO-releasing leachablematerials. At 35 minutes, the tube was reinserted into the reactorvessel and NO was released over the next 2 hour period, producing NO ata rate of 5.3×10⁻¹¹ moles NO/mg resin/min.

FIG. 3 illustrates a cut-away view of one embodiment of a device fordelivering nitric oxide to a flowing perfusate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a novel class of polymeric materialsthat contain the —[N(O)NO]⁻ functional group bound to a carbon atom. TheC-based polymeric diazeniumdiolates of the present invention are usefulfor a number of reasons. For example, C-based polymericdiazeniumdiolates are advantageous as pharmacological agents, researchtools, or as part of a medical device due to their ability to releasepharmacologically relevant levels of nitric oxide under physiologicalconditions without the possibility of forming potent nitrosaminecarcinogens. The C-based polymeric diazeniumdiolates of the presentinvention are insoluble. This property gives this class of materials anumber of uses and advantages, including but not limited to: 1) deliveryof NO to static or flowing aqueous solutions; and 2) the ability toremove the polymer from a solution or suspension by filtration orseparation after it has delivered nitric oxide. Furthermore, theinsoluble polymeric nature of the material allows embodiments of thisinvention to be used to construct NO-releasing medical devices.

In Formulas 1, 2, and 3, R¹ may not be represented by an imidate orthioimidate. R¹ may be represented by, but is not limited to an electronwithdrawing group such as but not limited to a cyano group; an ethergroup, such as, but not limited to —OCH₃, —OC₂H₅, and —OSi(CH₃)₃; atertiary amine; or a thioether, such as, but not limited to, —SC₂H₅, and—SPh (where the Ph is substituted or unsubstituted). The R¹ group mayalso be a amine, such as, but not limited to, —N(C₂H₅)₂, and in apreferred embodiment is an amine other than an enamine.

The R² group in Formulas 1, 2, and 3 may be a countercation or acovalently bound protecting group. In embodiments where the R² group isa countercation, the R² group may be any countercation, pharmaceuticallyacceptable or not, including but not limited to alkali metals such assodium, potassium, lithium; Group IIa metals such as calcium andmagnesium; transition metals such as iron, copper, and zinc, as well asthe other Group Ib elements such as silver and gold. Otherpharmaceutically acceptable countercations that may be used include butare not limited to ammonium, other quaternary amines such as but notlimited to choline, benzalkonium ion derivatives. As understood by thoseskilled in the art, the negatively charged diazeniumdiolate group mustbe counterbalanced with equivalent positive charge. Thus, referring toFormula 1, the valence number of the countercation or countercations(R²) must match the stoichiometric number of diazeniumdiolate groups,both represented by y. In embodiments where more than onediazeniumdiolate is bound to the benzylic carbon, and R² is monovalent,R² can be the same cation or different cations.

R² can also be any inorganic or organic group covalently bound to theO²—oxygen of the diazeniumdiolate functional group including but notlimited to substituted or unsubstituted aryl groups, as well as asulfonyl, glycosyl, acyl, alkyl or olefinic group. The alkyl andolefinic group can be a straight chain, branched chain or substitutedchain. R² may be a saturated alkyl, such as, methyl or ethyl or anunsaturated alkyl (such as allyl or vinyl). Vinyl protecteddiazeniumdiolates are known to be metabolically activated by cytochromeP-450. R² may be a functionalized alkyl, such as, but not limited to,2-bromoethyl, 2-hydroxypropyl, 2-hydroxyethyl orS-acetyl-2-mercaptoethyl. The latter example is an esterase sensitiveprotecting group. The former examples provide a chemical functionalgroup handle. Such strategies have been successfully employed to linkpeptides to the diazeniumdiolate molecule. Hydrolysis may be prolongedby addition of the methoxymethyl protecting group. R² may be an arylgroup, such as 2,4-dinitrophenyl. This type of protecting group issensitive towards nucleophiles, such as glutathione and other thiols. Itis obvious to those skilled in the art that several different protectinggroups may be used, and/or the stoichiometry of the protecting groupaddition may be adjusted such that not all the diazeniumdiolate moietiesare protected with the same protecting group, or not all thediazeniumdiolate groups are protected at all. By using differentprotecting groups, or varying the stoichiometry of the protectinggroup(s) to diazeniumdiolate ratio, the practitioner may engineer therelease of NO to a desired rate.

R³ is a phenyl group. The phenyl group may be pendant from the polymerbackbone (as shown in Formula 2) or part of the polymer backbone (asshown in Formula 3). In non-polymeric embodiments R³ may be asubstituted or non-substituted phenyl group.

Any of a wide variety of polymers can be used in the context of thepresent invention. It is only necessary that the polymer selected isbiologically acceptable. Illustrative of the polymers suitable for usein the present invention and used as the “Polymer”, “Polymer¹”, or“Polymer²” (collectively “Polymer”) in the general formulas include, butare not limited to: polystyrene; poly(α-methylstyrene);poly(4-methylstyrene); polyvinyltoluene; polyvinyl stearate;polyvinylpyrrolidone; poly(4-vinylpyridine); poly(4-vinylphenol);poly(1-vinylnaphthalene); poly(2-vinylnaphthalene); poly(vinyl methylketone); poly(vinylidene fluoride); poly(vinylbenzyl chloride);polyvinyl alcohol; poly(vinyl acetate); poly(4-vinylbiphenyl);poly(9-vinylcarbazole); poly(2-vinylpyridine); poly(4-vinylpyridine);polybutadiene; polybutene; poly(butyl acrylate); poly(1,4-butyleneadipate); poly(1,4-butylene terephthalate); poly(ethyleneterephthalate); poly(ethylene succinate); poly(butyl methacrylate);poly(ethylene oxide); polychloroprene; polyethylene;polytetrafluoroethylene; polyvinylchloride; polypropylene;polydimethylsiloxane; polyacrylonitrile; polyaniline; polysulfone;polyethylene glycol; polypropylene glycol; polyacrylic acid;polyallylamine; poly(benzyl methacrylate); derivatized polyolefins suchas polyethylenimine; poly(ethyl methacrylate); polyisobutylene;poly(isobutyl methacrylate); polyisoprene; poly(DL-lactide); poly(methylmethacrylate); polypyrrole; poly(carbonate urethane); poly[di(ethyleneglycol) adipate]; polyepichlorohydrin; phenolic resins (novolacs andresoles); poly(ethyl acrylate); and combinations thereof includinggrafts and copolymerizations.

Polymer may also be represented by a styrenic resin, including, but notlimited to: acrylonitrile butadiene styrene terpolymer;acrylonitrile-chlorinated polyethylene-styrene terpolymer; acrylicstyrene acrylonitrile terpolymer; styrene acrylonitrile copolymers;olefin modified styrene acrylonitrile copolymers; and styrene butadienecopolymers.

Furthermore, Polymer may be represented by a polyamide, including, butnot limited to: polyacrylamide; poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone];poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/poly(butylene adipate)];poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/poly(ethylene glycol-co-propyleneglycol)/polycaprolactone]; poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/polytetrahydrofuran]; terephthalic acidand isophthalic acid derivatives of aromatic polyamides (e.g. Nylon 6Tand Nylon 61, respectively);poly(imino-1,4-phenyleneiminocarbonyl-1,4-phenylenecarbonyl);poly(m-phenylene isophthalamide); poly(p-benzamide);poly(trimethylhexamethylene terephthalatamide); poly-m-xylyeneadipamide; poly(meta-phenylene isophthalamide) (e.g. Nomex); copolymersand combinations thereof; and the like.

Also, Polymer may be represented by polymers including, but not limitedto: polyesters; polyarylates; polycarbonates; polyetherimides;polyimides (e.g. Kapton); and polyketones (polyether ketone, polyetherether ketone, polyether ether ketone, and the like); copolymers andcombinations thereof; and the like.

Polymer may be represented by a biodegradable polymer including, but notlimited to: polylactic acid; polyglycolic acid; poly(ε-caprolactone);copolymers; biopolymers, such as peptides, proteins, oligonucleotides,antibodies and nucleic acids, starburst dendrimers; and combinationsthereof.

Polymer may also be represented by silane and siloxane mono- andmultilayers.

Embodiments with Pendant Phenyl Groups

The pendant phenyl ring from the polymer may have substitutions. Thesubstituted phenyl may be substituted with any moiety that does notinterfere with the NO-releasing properties of the compound and maintainsa covalent bond to the polymer backbone. Appropriate moieties include,but are not limited to, aliphatic, aromatic and non-aromatic cyclicgroups. Aliphatic moieties are comprised of carbon and hydrogen but mayalso contain a halogen, nitrogen, oxygen, sulfur, or phosphorus.Aromatic cyclic groups are comprised of at least one aromatic ring.Non-aromatic cyclic groups are comprised of a ring structure with noaromatic rings. The phenyl ring may also be incorporated in multi ringsystems examples of which include, but are not limited to, acridine,anthracene, benzazapine, benzodioxepin, benzothiadiazapine, carbazole,cinnoline, fluorescein, isoquinoline, naphthalene, phenanthrene,phenanthradine, phenazine, phthalazine, quinoline, quinoxaline, andother like polycyclic aromatic hydrocarbons. Additional moieties thatcan be substituted on the phenyl ring include, but are not limited to,mono- or di-substituted amino, unsubstituted amino, ammonium, alkoxy,acetoxy, aryloxy, acetamide, aldehyde, benzyl, cyano, nitro, thio,sulfonic, vinyl, carboxyl, nitroso, trihalosilane, trialkylsilane,trialkylsiloxane, trialkoxysilane, diazeniumdiolate, hydroxyl, halogen,trihalomethyl, ketone, benzyl, and alkylthio.

Polymers according to the present invention may be derived fromcommercially available chloromethylated polystyrene. Alternatively,chloromethylated polystyrene may be synthesized in a number of ways,including, but not limited to: utilizing chloromethyl alkyl ethers inthe presence of Lewis acid catalysts (Merrifield, 1963); oxidation ofpoly(4-methylstyrene) using cobalt(III) acetate in the presence oflithium chloride (Sheng and Stover, 1997); or treatment ofp-methylstyrene with sodium hypochlorite solution in the presence ofphase transfer catalysts (Mohanraj and Ford, 1986; Le Carre et al.,2000).

In one preferred embodiment of the present invention, using Formula 2, apolymer may be synthesized in a two-step procedure as outlined inScheme 1. In the first step (1), chloromethylated polystyrene is treatedusing methods known in the art to replace the —Cl atom with anucleophilic substituent. It is desirable that the nucleophilicsubstituent activates the benzylic carbon protons for the introductionof diazeniumdiolate functional groups. In a preferred embodiment of thisinvention, the atom replacing the —Cl atom of the chloromethylatedpolystyrene is an electronegative heteroatom. It is preferred that thenucleophilic group replacing the —Cl atom is electron withdrawing. It ismost preferred that the substituent be a cyano group. Additionalpreferred substituents may be selected from a group that includes —OR,—NR₁R₂, and —SR. The —OR group may be, but is not limited to, —OCH₃,—OC₂H₅, and —OSi(CH₃)₃. The replacing group may be a thiol group, suchas, but not limited to, —SC₂H₅, and —SPh (where the Ph group issubstituted or unsubstituted). The replacing group may also be a amine,such as, but not limited to, —N(C₂H₅)₂.

The second step (2) in Scheme 1 requires treatment of the polymer with abase in the presence of NO gas. The solvent for the reaction should notreact with NO in the presence of a base (e.g. acetonitrile or ethanol).It is preferable that the selected solvent should swell the polystyrene.Suitable solvents include, but are not limited to, THF and DMF. Suitablebases include, but are not limited to, sodium methoxide and sodiumtrimethylsilanolate. In accordance with the method of the invention theresulting resin derived from chloromethylated polystyrene followingthese procedures will contain multiple —[N(O)NO]⁻ functional groupswhich spontaneously release NO in aqueous media. The R² substituentreferred to in the general Formulas and Scheme 1 represents apharmaceutically acceptable counterion, hydrolysable group, orenzymatically-activated hydrolysable group as described above.

Embodiments Using Silane/Siloxane Polymers

In another preferred embodiment of the present invention, using Formula2 where polymer is represented by a siloxane, a NO-releasing siloxanepolymer may be synthesized in a similar procedure as outlined in Scheme1 where the material is first coated with the silane/siloxane and thenmodified to an NO-releasing agent. A general description of surfacepreparation and silane/siloxane deposition is described below.

Surface Preparation

For the process of creating an embodiment of the present invention, anNO-releasing coating that is covalently bound to the substrate surface,it is critical to have a surface that presents pendant hydroxyl groups.As known to those skilled in the art, many surfaces can be easilymodified (oxidized) to contain hydroxyl groups pendant to the surface.Such surface treatments include but are not limited to soaking inconcentrated NaOH or KOH, or exposure to concentrated solutions ofhydrogen peroxide (Srinivasan, 1988; Endo, 1995; Yoshida, 1999;Fitzhugh, U.S. Pat. No. 6,270,779; Kern, 1995.). The examples sectionwill describe specific methodology for producing surface hydroxylgroups.

Once the surface is in the appropriate chemical form, the siloxane(s)coating can be deposited. For embodiments requiring dense, horizontalmonolayers, trichlorosiloxane derivatives are preferred, and for thickervertical coatings, alkoxysiloxane derivatives are preferred. Eachembodiment requires a specific chemical methodology.

Formation of Monolayers

In embodiments of the present invention where dense monolayers ofC-based diazeniumdiolate coatings are preferred, deposition of thecommercially available 4-cyanomethylphenyl triethoxysilane,4-chloromethylphenyl trichlorosilane, or any trichlorosilane thatcontains a pendant methylphenyl group where the benzylic carbon can besubstituted with any group which allows for substitution ofdiazeniumdiolate functional groups on the benzylic carbon atom ispreferred. For embodiments where the cyano-substituted benzylic carbonis desired, it is preferred to deposit the commercially available4-cyanomethylphenyl triethoxysilane on the surface. For all otherembodiments, it is preferred to deposit the commercially available4-chloromethylphenyl trichlorosilane onto the surface, and, at asubsequent step, substitute the chloro atom for the desired substituentusing the appropriate nucleophile as described in the “Substituting aNucleophile” section below. This method eliminates the need forpotentially complicated synthesis of trichlorosiloxane derivatives withthe desired benzylic carbon substituent. It should be noted that it ispossible to use a trialkoxysilane under similar conditions to produce amonolayer (Bierbaum, 1995), however the high reactivity of thetrichlorosiloxane derivatives to what is a very minimal amount ofsurface water causes the trichloro derivatives to be preferred formonolayer applications.

Typically, the trichlorosilanes are deposited using anhydrousconditions, using a 0.1-3% trichlorosilane solution in a hydrocarbonsolvent such as toluene or hexadecane under an inert atmosphere. Theapplication of the trichlorosilane solution can be applied to thedesired surface under anhydrous conditions and an inert atmosphere via avariety of methods including but not limited to dipping, vapordeposition, spray coating, flow coating, brushing and other methodsknown to those skilled in the art. The polymerization is usuallycomplete from 1 to 24 hours. The material is then rinsed with ahydrocarbon solvent, heat cured at 110° C. for 20 to 60 min to formcovalent bonds with the surface hydroxyls as described below, andprepared for further use. While not wishing to be bound to anyparticular theory, the monolayer is formed as follows. The waternecessary for the polymerization of the trichlorosilane derivatives isprovided by the intrinsic water found on the surface of most substrates.Because this inherent surface water is the only available water to drivethe polymerization reaction, the polymerization of the silanederivatives can only occur at the surface of the material. Thus, thelocalization of water to the surface limits the polymerization to asurface monolayer and only trichlorosilane molecules contacting thesolid surface are hydrolyzed, producing a closely packed monolayer. Toomuch water, such as where rigorous anhydrous conditions in the solventare not observed, will lead to rapid polymerization of the silanes,possibly before they have even had a chance to deposit on the substratesurface (Silberzan, 1991). In comparison, hydrolysis of alkoxysilanes in95% alcoholic solutions results in significant oligomerization of thesilanes before the substrate to be coated is introduced into thesolution. Numerous reports support this scheme (Ulman, 1996; Sagiv,1980; Wasserman, 1989; Bierbaum, 1995).

It should be noted, and is known by those skilled in the art, that thisprocess of monolayer deposition can be repeated using multipleapplications of trichlorosilane derivatives, resulting in the ability tobuild many subsequent monolayers (Tillman, 1989).

Formation of Three Dimensional Networks

In embodiments of the present invention where thicker, more verticallypolymerized C-based diazeniumdiolate coatings are preferred, thealkoxysilane class of siloxane is preferred. The appropriatealkoxysilanes, such as but not limited to cyanomethylphenyl alkoxysilanederivatives, chloromethylphenyl alkoxysilane derivatives, or anyalkoxysilane derivative capable of permanently entrapping achloromethylphenyl or cyanomethylphenyl group within its matrix ispreferred. Generally, a 95% ethanol 5% water solution is adjusted to pH5±0.5 with acetic acid and the appropriate alkoxy silane is added to aconcentration between 1 and 10% (v/v). During the next several minutes,the alkoxysilane derivatives will undergo hydrolysis to form silanolswhich will condense to form oligomers. At this point the substrate canbe dipped, or otherwise coated according to methods known to thoseskilled in the art. While not wishing to be bound to any particulartheory, the silanols condense into larger oligomers which hydrogen bondto the surface hydroxyls of the substrate and can reach out like ‘hairs’on the surface. The siloxane(s) continue to polymerize and form verticalmatrices. The duration of exposure of the substrate to the alkoxysilanederivative is generally proportional to the thickness of the coatingformed. At the desired time point, the coated material is rinsed withethanol, heat cured at 110° C. for 20 to 60 min if desired, and preparedfor further use.

The appropriate methylphenyl siloxane derivative may be used pure or inany fraction with other siloxane(s) to form the coating, as well as withother compatible polymers.

Once the desired siloxane coating has been deposited, the formation ofcovalent bonds between the coating and the oxidized substrate surfacecan be achieved. This is accomplished through the application of dryheat, typically but not exclusively at 110° C. for 20 to 60 min. Withoutbeing bound by any particular theory, under the conditions typical toapplying dry heat, the hydroxyl moieties in the siloxane coating thatare hydrogen bonded to the hydroxylated surface of a substrate willreact through a dehydration reaction and form strong covalentsilicon-oxygen bonds.

Substituting a Nucleophile

In the case where cyanomethylphenyl siloxanes are used in the coatingstep, the addition of a nucleophile to the benzylic carbon is notnecessary, as the cyano group is an excellent activating group. Use ofcyanomethylphenyl siloxanes allows the practitioner to go directly tothe diazeniumdiolation step. If a chloromethyphenyl siloxane or otherchloromethyphenyl derivative is used, or the practitioner desires tochange the nucleophile, thereby changing the characteristics of thediazeniumdiolate group and thus altering the rate of release of NO fromthe coating, the chloro group must be exchanged with a nucleophile thatallows for the introduction of the diazeniumdiolate group as describedabove. This step is performed as follows: The coated substrate isimmersed in a solution of DMF containing a catalytic amount of potassiumiodide and the nucleophile of choice. The solution is heated to 80° C.for up to 24 hours. During this time the substitution reaction occurs.The substrate is then removed from the solvent, washed with fresh DMFand blown dry with nitrogen or left in air to dry.

Diazeniumdiolation Step

Once the appropriate nucleophile is added to the benzylic carbon of theappropriate siloxane derivative, the coated material is placed in a Parrpressure vessel containing a solvent such as THF, DMF or MeOH. Asterically hindered base such as sodium trimethylsilanolate is added.The choice of base is important because the silicon-oxygen bonds of thesiloxane network are sensitive to aggressive nucleophiles such ashydroxides and alkoxides. The vessel is purged of atmosphere with aninert gas and pressure checked before exposure to several atmospherespure NO gas. After 1 to 3 days, the coated materials are removed, washedand dried in air before storage under argon at 4° C.

Embodiments with Polymeric Backbone Comprising Phenyl Groups

The polymeric NO releasing resin described in various examples above hasthe —[N(O)NO]⁻ functional groups pendant to the polymeric backbone. Thepresent invention also provides methods to modify any phenyl ring foundin the backbone of the polymer. Thus, other means to introduce thenucleophile to obtain the molecular arrangement shown in Formula 1 areconsidered within the scope of the present invention.

Considering Formula 3, Polymer¹ and Polymer² may be equivalent ordifferent from each other, and may include but not be limited to:polybutylene terephthalate; polytrimethylene terephthalate; andpolycyclohexylenedimethylene terephthalate. In addition, aramides (aclass of polymers in the nylon family synthesized from the reaction ofterephthalic acid and a diamine) may also be represented by Polymer¹ orPolymer². Examples of such aramides include, but are not limited to,poly(p-phenylene terephthalamide) and poly(m-phenylene isophthalamide).As in other embodiments of this invention described above, it isdesirable that the nucleophilic substituent activates the benzyliccarbon protons for the introduction of diazeniumdiolate functionalgroups.

In a preferred embodiment, the atom replacing the —Cl atom of thechloromethylated polystyrene is an electronegative heteroatom. It ispreferred that the nucleophilic group replacing the —Cl atom is electronwithdrawing. Preferred substituents for R¹ may be represented by, butare not limited to: a cyano group; an ether group, such as, but notlimited to —OCH₃, —OC₂H₅, and —OSi(CH₃)₃; a tertiary amine; and athioether, such as, but not limited to, —SC₂H₅, and —SPh (where the Phgroup can be substituted or unsubstituted). The R¹ group may also be aamine such as, but not limited to, —N(C₂, H₅)₂.

Polyethylene terephthalate (PET) is used in an exemplary embodiment ofthe present invention, where Polymer¹ and Polymer² in Formula 3represent the repeating ethylene-terephthalate structure. Condensationof terephthalic acid and a diol such as ethylene glycol results in thepolyester. Other examples of polyesters can be produced by variation ofthe diol. Such polyesters may be transformed into NO-releasing materialsin a four step process.

By way of example and not in limitation, as shown in Scheme 2, thearomatic ring contained in a polymer of PET may be treated withformaldehyde and acetic acid to produce a benzyl alcohol (Yang, 2000).Treatment with tosyl chloride introduces an effective leaving group ontothe polymer. Further treatment with a nucleophile of choice willdisplace the tosylate and provide the necessary structure forintroduction of the —[N(O)NO]⁻ functional group. Therefore, it should beapparent to one of ordinary skill in the art that there may be a widevariety of polymers containing an aromatic phenyl group which may bemodified to contain the necessary chemical structure for transformationinto a carbon-based diazeniumdiolate through the teachings of thepresent invention.

General Chemistry and Strategies to Control Release of NO

Without restraint to any one theory, the importance of the benzylicstructure (methylphenyl group) to the invention is threefold. First, thebenzylic carbon has relatively acidic protons and the choice ofnucleophile should increase the acidity of the benzylic protons suchthat a base can easily extract a proton. Exposure of benzylic compoundsto NO gas in the absence of base is not known to produce thediazeniumdiolate functional group. Secondly, the aromatic ring resonancestabilizes the carbanion formed by extraction of a proton by base. Thestabilized carbanion allows for the reaction of the carbanion with NO,to produce a radical carbon center and nitroxyl anion (NO⁻). Furtherreaction of the radical carbon center with NO or NO dimer produces thediazeniumdiolate functional group. The anionic diazeniumdiolatefunctional group enhances the acidity of the last benzylic proton andallows an additional diazeniumdiolate group to be added to the carbon.In this manner, up to three diazeniumdiolate functional groups areintroduced into the polymer via the benzylic carbon. Thirdly, thepresence of resonant electrons in the aromatic ring helps promotespontaneous decomposition of the —[N(O)NO]⁻ group in aqueous media.Other bisdiazeniumdiolates, namely methylene bisdiazeniumdiolate[H₂C(N₂O₂Na)₂] lack resonant electronic forces that participate in thedecomposition process and thus show remarkable stability (inability torelease NO) in solution (Traube, 1898).

In addition to their advantage of releasing NO under physiologicalconditions without forming nitrosamine carcinogens, the degree and rateof NO release of these polymeric materials may be engineered usingseveral types of manipulations. FIGS. 1 and 2 show the NO releaseprofiles of two different C-based NO releasing head groups attached tomethyl polystyrene. The structural differences in the NO-releasingheadgroup were achieved by changing the nucleophile that results in theR¹ substituent. The release profile in FIG. 1 is the result of acyano-modified (R¹) benzylic carbon and FIG. 2 shows an ethoxy-modified(R¹) benzylic carbon. Examination of the Figures indicates thecyano-modified polymer exhibits a rapid release profile, whereas theethoxy-modified polymer exhibits a prolonged but less robust release ofNO. Several more examples of the results of manipulation of R¹ on NOrelease properties are described in the Examples. It should be apparentto one skilled in the art that a contiguous polymer may contain morethan one type of nucleophilic substituent. As shown in Scheme 3,chloromethylated polystyrene cross-linked with divinylbenzene can bemodified with two different nucleophiles, R¹ _(a) and R¹ _(b), toproduce two different types of NO-donor moieties. The ability to controlthe release rate of NO through manipulation of R¹ allows for preciseengineering of the release of NO from the polymer on a macro scale.

Another preferred way of reaching the desired amount and rate of NOrelease on a macro scale is to blend two or more of the individuallysynthesized polymers together to achieve the desired rate of NO releasefrom the polymer. This method has the advantage over manipulating R¹ inthe NO-releasing headgroups of a single polymer because it eliminatesthe need for stoichiometric control of the synthetic chemistry toachieve the desired release rate. However, this method may not be easilyamenable to micro- and nano-scale applications.

An additional way to affect the rate and degree of NO release from thepolymer, one which especially holds relevant for the polystyrene-basedpolymers, is to vary the degree of cross-linking of the polymer.Generally, a lesser degree of cross-linking provides a more porouspolymeric structure. While this does not change the degree ofnucleophilic substitution and diazeniumdiolation, it provides a morerapid and greater degree of NO release from the polymer because theactive NO-releasing sites are more accessible to the aqueous solvent.Increasing the polymer cross-linking decreases the porosity of thepolymer, which serves to inhibit aqueous solvent access. Highlycross-linked polymers release NO for longer periods of time (see, forexample, U.S. Patent Application Pub. No.: US 2003/0077243 A1). Thus,various rates of NO-release may be obtained by controlling the access ofaqueous solution to the —[N(O)NO]⁻ functional groups through the degreeof cross-linking of the polymer.

The C-based diazeniumdiolate polymer of the present invention is also animprovement over the prior art in terms of time of synthesis and amountof NO generated. For example, according to the teachings of U.S. Pat.No. 5,405,919, a polyamine was linked to chloromethylated polystyreneand a slurry of the aminopolystyrene in acetonitrile was subsequentlyexposed to NO to produce a N-based diazeniumdiolate. However, such aN-based diazeniumdiolate required a week to synthesize and produced verylow levels of NO under physiological conditions which is not useful formany applications. The method of the present invention utilizes asuitable solvent to swell the resin and adding potassium iodide as acatalyst to accelerate the nucleophilic substitution reaction, which isa significant improvement over the reaction time (2 days versus 8 days)and NO-release levels (ppm NO versus very low levels) when compared tothat disclosed in U.S. Pat. No. 5,405,919.

Polymers that release NO are desirable for providing localized fluxes ofNO at the specific target sites. The NO may be localized in vivo, usedin ex vivo applications of cells, tissues, and organs, or as in vitroreagents. In applications where NO is applied to cells in culture, theuse of polymeric materials provide a distinct advantage in that they areeasily separated from the cell suspension due to their size and/ordensity.

Polymeric forms of diazeniumdiolate nitric oxide donors can be used toprovide localized delivery of nitric oxide, and therefore are useful indevices such as stents, prostheses, implants, and a variety of othermedical devices. Polymeric materials may also be used in in vitro and exvivo biomedical applications.

Use of the Present Invention in Coatings for Medical Devices

The present invention provides methods for a novel class of coatings inwhich NO-releasing carbon-based diazeniumdiolates may be covalentlylinked to a surface, whereby the release of NO imparts increasedbiocompatibility or other beneficial properties to the coated surface.In order for NO to be therapeutic it is most preferable that it bedelivered/produced at the site of interest. The polymers describedherein have the potential to generate NO temporally and spatially at thedesired area of interest. Thus, a medical device comprised of theNO-releasing polymers may provide a localized flux of NO without anydeleterious systemic effects such as hypotension. The beneficialphysiological properties of NO may be targeted directly at desired siteof application. The structural and physical characteristics of theNO-releasing polymers in the present invention may be manipulated tosuit the treatment of the biological disorder. The polymers may take theform of a device such as an arterial stent, vascular graft, patch, orimplant. The NO-releasing polymers may also be microencapsulated orenteric coated for ingestion. In addition, the NO-releasing polymers ofthe present invention may be incorporated into other polymericstructures by co-polymerization, precipitation or deposition aspracticed by those skilled in the art.

As one skilled in the art would appreciate, exemplary embodiments of thepresent invention find utility in a wide variety of applicationsdepending upon the physiological disorder. One possible preferredapplication for this class of coatings would be in medical devices wherethe surface can be comprised of but is not limited to metals includingtitanium, alloys of titanium including Ti₆Al₄V and nitinol, niobium,molybdenum, chromium, aluminum, nickel, copper, gold, silver, platinum,vanadium, all alloys and combinations thereof, all varieties ofstainless steel including surgical grade, and any metal capable offorming surface oxide groups; silicates including but not limited toglass, fused silica glass, 96% silica glass, aluminosilicate glass,borosilicate glass, lead glass, soda lime glass; polymers comprised ofbut not limited to silastic, hydroxylated polyolefins, or any plastic orpolymeric material with pendant surface hydroxyl groups, includingbiopolymers.

Vascular Stents

Each year in the U.S. about 700,000 patients suffering from coronaryatherosclerosis, blockage or narrowing of the arteries to the heart,undergo percutaneous transluminal coronary angioplasty (PTCA) as a meansto return normal circulation to the heart. This procedure involves theinflation of a balloon catheter in the narrowed area of the coronaryartery thus enlarging the diameter and increasing the blood flow to theaffected area. However, approximately 30-50% of the time, the arterialocclusion returns in a process termed restenosis. A preventive measurefollowing PTCA is the deployment of a vascular stent to act as a supportin the artery. Despite this treatment, restenosis still occurs in 15-25%of patients receiving stents and additional treatment is required.

The current state of the art vascular stents are designed to eluteanti-proliferative medications such as sirolimus as a means to inhibitrestenosis. However, these drugs are not antithrombotic and patientshave developed life threatening blood clots. Furthermore, theanti-proliferative drugs inhibits the growth of vascular endothelialcells, which are beneficial to the post angioplasty healing process. Theanti-proliferative drug-eluting stent exemplifies a fundamental problemunderlying the development of drug-eluting stents. There is no singledrug that stands out as an effective treatment for this disease.

An alternative approach towards treating restenosis is to incorporate anatural product that inhibits platelet aggregation, prevents smoothmuscle cell proliferation and promotes re-endothelialization of theinjured vessel and endothelialization of the stent surface. Nitric oxide(NO) can perform these physiological functions. A vascular stent can becoated with the present invention to elute therapeutic amounts of NOwhich would accelerate the healing process following PTCA stentdeployment thus improving patient outcome over the current state of theart drug eluting stents.

By way of example and not limitation, a cardiovascular stent comprisedof or coated with the NO-releasing polymers of the present inventionwill possess the ability to resist platelet adhesion, prevent plateletaggregation, inhibit vascular smooth muscle cell proliferation(Mooradian et al., 1995), and stimulate the proliferation of vascularendothelial cells. The current state of the art anti-proliferativeeluting stents do not inhibit blood clot formation. Patients receivingthese stents must maintain a 3-month regimen of anti-clottingmedication. Recent reports disclose the detection of blood clots indozens of patients who have received this type of stent (Neergaard,2003). One skilled in the art can utilize a coating that releases boththe anti-proliferative drug and NO simultaneously.

The proliferation of endothelial cells (ECs) by NO is of great interestbecause it is the first step towards neovascularization (Ausprunk,1977). If NO can stimulate EC proliferation then an inserted medicaldevice such as a vascular stent or graft modified with a NO-releasingcoating of the present invention might be able to promote overgrowth ofthe device with endothelial tissue. In this way, blood contact with thedevice will move from the NO-releasing coating to a natural cellularlayer. Recently, a group has genetically engineered endothelial cells toover-express endothelial nitric oxide synthase (eNOS) in an attempt toenhance the EC retention on a vascular graft (Kader, 2000).

Other Vascular Devices

The various beneficial effects of NO in the cardiovascular system can befurther exploited using the present invention. One skilled in the artwill realize that the anti-platelet effect will be useful when appliedas a coating to vascular grafts or when the polymers of the presentinvention are formed into vascular grafts. The NO-releasing polymer willgive off sufficient NO for sufficient duration to eliminate bloodclotting events from occurring until the graft can be overgrown withendothelial cells.

One skilled in the art will also realize that polymers from the presentinvention can be used in extracorporeal membrane oxygenation circuits(ECMO), more commonly known as a “heart/lung machine.” A majorcomplication of this procedure is the loss of platelets due to adhesionalong the inner surface of the tubing used to form the extracorporealcircuit. A thromboresistant surface made from N-based diazeniumdiolatesmall molecules embedded in a polymer matrix reduced the loss ofplatelets in a rabbit model of ECMO (Annich et al., 2000). However, thepolymer in the study has the disadvantages associated with N-baseddiazeniumdiolate polymers (i.e., potential carcinogen). Polymers of thepresent invention do not have the associated toxic potential of theN-based diazeniumdiolates.

Another beneficial application of the present invention is for patientsundergoing hemodialysis. Application of the present invention to shuntsused for hemodialysis, extracorporeal tubing, and the dialysis membraneitself can be used to decrease the adhesion of platelets to thesurfaces, resulting in increased circulating platelets in the patient.

Additional applications of the present invention include but are notlimited to increasing the patency of percutaneous needles, increasingthe thromboresistance of indwelling sensors and surgical tools,engineering the formation of new blood vessels, treating hypertension,and other applications were localized therapeutic levels of NO would bebeneficial to the patient.

Indwelling Catheters

An endemic problem associated with hospitalization is manifested in thenumber of infections and deaths directly related to inserted medicaldevices such as catheters, shunts, and probes. It is estimated that upto 20,000 deaths occur each year due to infection acquired from vascularcatheterization. The inserted medical device provides direct access intothe body for advantageous skin microorganisms. These bacteria adhere toand colonize upon the inserted device and in the process form anantibiotic resistant matrix known as a biofilm. As the biofilm grows,planktonic cells can break free and spread the infection further intothe patient. In order to prevent infection, the inserted medical devicemust prevent the biofilm formation. This can be done by killing thebacteria before they can colonize the medical device or prevent theadhesion of bacteria to the device such that a biofilm cannot form.

It is well known that NO can prevent blood platelets from adhering tovarious surfaces and NO has antimicrobial properties. A recent reportdemonstrates that NO can also inhibit bacterial adhesion (Nablo et al,2001). Polyaminosiloxanes were deposited on glass slides and derivatizedinto NO donors. P. aeruginosa adhesion was inhibited in a dose dependentmanner by the NO-releasing sol-gels. This early report strongly suggeststhat bacterial adhesion can be influenced by surfaces designed torelease NO. Therefore, catheters coated with NO-releasing polymers ofthe present invention may inhibit biofilm formation and improve patienthealth care.

Contact Lens Cases

Contact lens-related eye infections impact millions of people yearly.Standard guidelines for lens care can minimize eye infection, but it hasbeen shown that only about 50% of lens wearers adhere to appropriateguidelines. Among contact lens wearers that do follow the recommendedguidelines, lens-related infections still occur. During usual use andstorage procedures, microorganisms adhere to contact lenses. Daily lenscleaning removes most of these microorganisms; however, microbes canestablish biofilms on lenses. Often such biofilms are not satisfactorilyremoved despite disinfection and cleaning with systems currentlyavailable. In many cases the source of the microorganisms is the lenscase (McLaughlin et al. 1998). Even for non-symptomatic lens wearers,the lens case contains bacterial biofilms, and this source most likelyserves as an important contamination route for lenses, despite the useof disinfectants and cleaning solutions (McLaughlin et al. 1998). Inaddition, biofilms formed by pathogenic organisms are of increasingclinical importance due to their resistance to antibiotics and hostimmune responses, as well as their ability to develop on indwellingmedical devices.

Use of the Present Invention in the Manufacture of Medical Devices

In addition to the ability to coat medical devices, the presentinvention also provides a method to manufacture devices or components ofdevices using NO-releasing polymers. Many of the exemplary embodimentsof the present invention, use of such starting materials as, but notlimited to, PET, PS, siloxane-based polymers, all of which can be usedto manufacture entire medical devices or components thereof.

-   -   NO-releasing polymers of the present invention may be        synthesized and extruded, molded, injection molded, blow molded,        thermoformed or otherwise formed into complete devices or        components thereof using methods known to those of skill in the        art to produce solid devices or device components that release        NO and comprise a medical device.

In an alternative method, the device or device components aremanufactured using an appropriate non-NO-releasing polymer, andmodifying the device or device components to release NO as described inExample 8.

Use in Platelet Storage Applications

One non-limiting example of the utility of NO-releasing polymers is inthe ex vivo inhibition of platelets. Nitric oxide has been shown to be apotent inhibitor of platelet aggregation (Moncada et al., 1991).Application of NO to platelets also results in a decreased intracellularcalcium response to agonists (Raulli, 1998) as well as otherintracellular processes dependent on calcium, such as release of granulecontents (Barrett et al., 1989). Example 12 shows the ability ofNO-releasing polymers to inhibit agonist-induced platelet aggregation.

This ability of NO-releasing polymers to inhibit platelet activation exvivo may be of considerable utility in the treatment of Platelet StorageLesion (PSL). Platelet Storage Lesion is defined as the sum of thechanges that occur in platelets following their collection, preparation,and storage (Chrenoff, 1992), and is responsible for the loss ofplatelet functionality that increases with increased duration ofstorage. These changes include cytoskeletal and surface antigenstructural changes, release of dense and alpha granule contents, releaseof lysosomal contents, loss of membrane integrity, and defects ofmetabolism (Klinger, 1996). The mechanism(s) that cause PSL are poorlyunderstood, but a general consensus is that PSL is related (at leastpartially) to the results of platelet activation during the storageperiod (Snyder (ed), 1992). Because NO is a known inhibitor of plateletactivation (Moncada et al., 1991) and activation of storage granules(Barrett et al., 1989), treatment of stored platelets with NO-releasingagents may reduce the degree of PSL, resulting in an increasedactivatable platelet count, e.g., platelets that have their alpha anddense granules intact, decreased cellular debris, decreased autocoidconcentration of the storage plasma, and decreased morphological changesthat may affect platelet performance.

One skilled in the art can devise a number of ways to treat storedplatelets with NO-releasing polymers. An exemplary embodiment of thepresent invention uses a carbon-based nitric oxide-releasing polymerthat is manufactured pre-loaded within the blood storage compartment.The polymer should be of appropriate quantity and release rate topartially or completely inhibit platelet activation for a specifiedamount of platelet-rich plasma (PRP), platelet concentrate (PC),apheresed platelets (APP), or other platelet product that would betraditionally stored. The polymer should release inhibitory levels ofnitric oxide for sufficient duration to cover the entire predictedduration period for the platelet product, although paradigms can beenvisioned where the inhibitory flux of nitric oxide need not be presentfor the entire duration of storage.

The NO-releasing polymer may be a single entity or a blend of polymersdesigned to reach an optimized release rate and duration of NO release.Furthermore, the polymer may be designed to maximize its surface area,without interfering with platelet agitation within the platelet storagecontainer. Also, the polymer may be anchored to the storage container,free, or contained within a permeable or semi-permeable membranecomprised of any material that is compatible with blood cells and bloodplasma. Free polymer embodiments should be of an appropriate size andshape so as not to enter or clog the exit port that delivers the bloodproduct to the recipient. Preferred embodiments would use, but not belimited to, polymers comprised of pendant carbon-based diazeniumdiolategroups. One skilled in the art would appreciate that NO-releasingpolymers could be part of a complete manufactured system for plateletstorage as described in U.S. Provisional Patent Application No.60/471,724, Raulli et al., Systems and Methods for Pathogen Reduction inBlood Products.

The use of NO-releasing polymers of the present invention may also beuseful in other applications as a platelet inhibitor. It is well knownthat exposure of human platelets to cold temperatures results in a“cold-induced” activation characterized by an immediate rise in plateletintracellular calcium levels (Oliver et al. 1999), and changes inmorphology (Winokur and Hartwig, 1995). Recent studies describe a methodto freeze-dry platelets (U.S. Pat. No. 5,827,741 Beattie et al.). Thefreeze-dried and reconstituted end product shows a 15 to 30% degradationof the viable platelet count (Wolkers et al. 2002). This may be due to acold-induced activation of platelets during the initial lyophilizationprocess, or the result of the thawing process. Exposure of the plateletsto NO-releasing polymers of the current invention prior to, during, orafter the lyophilization process may decrease or eliminate anycold-induced activation, and consequently may increase the viability ofthe freeze-dried platelets.

One skilled in the art can develop a variety of methods to incorporateC-based NO-releasing polymers of the present invention into methods forcooling, freezing, or freeze-drying platelet preparations. An exemplaryembodiment would be similar to those described above for inhibition ofstored platelets.

Use in Pathogen Reduction of Stored Human Platelets

It has been well established that nitric oxide can kill a variety ofbacterial, fungal and viral pathogens (DeGroote and Fang, 1995). Anexemplary embodiment of the current invention uses a nitricoxide-releasing polymer within the blood storage compartment thatdelivers sufficient levels of nitric oxide to reduce or eliminate viablemicrobes that may be contaminating the blood product (U.S. ProvisionalPatent Application No. 60/471,724, Raulli et al., Systems and Methodsfor Pathogen Reduction in Blood Products). Example 13 shows the abilityof NO-releasing polymers to pathogens in blood storage containers.

The polymer will release sufficient levels of nitric oxide at anappropriate rate and for sufficient duration to kill, inactivate, orretard the further growth of pathogens that contaminate the bloodproduct. Further, the polymer is comprised of material that iscompatible with blood cells and blood plasma. The polymer may also bedesigned to maximize its surface area, without interfering with plateletagitation in the case of a platelet storage container. In exemplaryembodiments, the polymer may be anchored to the storage container, freefloating, or contained within a permeable or semi-permeable membranecomprised of any material that is compatible with blood cells and bloodplasma. Free floating polymer embodiments should be of an appropriatesize and shape so as not to enter or clog the exit port that deliversthe blood product to the recipient. Preferred embodiments would usepolymers comprised of pendant C-based diazeniumdiolate groups.

Use in Perfusion of Organs and Tissues for Treatment of Ischemia,Preservation, and Transplantation

Nitric oxide has a potent and profound vasodilatory effect on mammalianblood vessels (Palmer et al., 1989). This pharmacological property, aswell as the chemical antioxidant property of NO (Espey et al., 2002)make NO useful in transplantation medicine. When applied to the perfusedorgan, nitric oxide, acting as a vasodilator, allows greater perfusionof the deep tissues of the organ, bringing oxygen and nutrients to thetissue. The deeper penetration of the perfusate also benefits the organin bringing more NO to the deep tissues, further enhancing theantioxidant ability of nitric oxide to prevent the oxidative damagetypical of reperfusion injury (Ferdinandy and Schultz, 2003; Wink etal., 1993 and references therein).

While numerous types of NO donors are effective as vasodilators, many,like sodium nitroprusside (Kowaluk et al., 1992) and nitrosothiols(Dicks et al, 1996), require metabolic activation making them lesspredictable. This is especially relevant given the fact that theperfusate may not contain the necessary factors required for activationof these compounds as compared to blood. In the case where tissue thiolsor metals are required for activation, the tissue itself may beunpredictably deficient or rich in these factors due to the effects ofischemia-related insult. Furthermore, these NO donors do not release thepreferred antioxidant species (NO.), or need additional factors such asCu to convert the release species to NO.. Finally, sodium nitroprusside(SNP), a common NO-releasing vasodilator, may give off cyanide afterdonating its NO. Such problems highlight some of the advantages ofexemplary embodiments of the current invention, namely that a devicegives off only NO and there are no spent donor molecules present in theperfusate.

The redox state of the released NO may be of particular interest. ManyNO donors such as SNP release nitrosonium ion (NO⁺) and some producenitroxyl ion (NO⁻). Both species have been shown to exacerbate theeffects of reactive oxygen species (ROS), which are the agents thatultimately cause the oxidative tissue damage in ischemia reperfusioninjury. The nitric oxide species released by the current invention isNO., which has been shown to counteract the ROS (Wink et al, 1996).

The ability of the polymers of the current invention to spontaneouslyand predictably release NO. represents an advantage over soluble NOdonors as potential treatments in the organ perfusion process. This“donorless” delivery of NO is possible because the NO-releasingheadgroup and the polymeric matrix to which it is attached remaininsoluble when in standing or flowing aqueous solutions, whilemaintaining their ability to release soluble NO into the solution. Inaddition to the inherent advantages of the current invention to delivera preferred antioxidant redox species of NO, this donorless approacheliminates the problem of circulating spent donor molecules.

Polymer(s) according to the present invention may be contained in anin-line device, whereby the flow of the perfusate through the devicereleases sufficient NO into the perfusate as to result in vasodilationof the organ vasculature and a neutralization of ROS in the perfusedorgan. An exemplary, but not limiting, embodiment is shown in FIG. 3.The device 300 includes a chamber 310, which could be cylindrical orother appropriate shape. Chamber 310 is closed at both ends usingfritted discs 330, which self-seal or seal with a gasket 340.Cylindrical chamber 310 is capped at each end by a funnel-shapedcollector 320 that channels fluid into a smaller nozzle 350, allowingfor facilitated attachment to a perfusion line 360 on each end of thedevice 300.

Contained within chamber 310 is a solid polymer 370, according to thepresent invention. Polymer 370 may be of any desirable shape, may beattached to the wall of chamber 310 or otherwise anchored, or freewithin the chamber. The size of polymer 370 may also vary. Howeverpolymer 370 must be of a size that will be easily contained by fitteddiscs 330 on either end of chamber 310. It is preferable that thedensity of polymer 370 within chamber 310 is such as to allow free flowof the perfusate through device 300.

Also, a mesh size of fritted discs 330 should also be optimized to allowfree flow of perfusate. One skilled in the art would appreciate that thesize of chamber 310 may have an impact on the levels of NO released intothe perfusate for any given flow rate, as the larger a chamber for agiven flow rate the longer the exposure of the perfusate to theNO-releasing polymer will be, resulting in more NO dissolved in theperfusate. One having ordinary skill in the art may appreciate that thesize, shape and geometry of the device 300 is merely exemplary and maybe readily changed and remain effective in releasing NO within aperfusate. All such variations are within the scope of the presentinvention.

Example 14 demonstrates an ability of polymers according to the presentinvention to deliver significant quantities of NO to buffers flowingthrough an in-line container comprised of a fritted chamber withNO-releasing polymer contained within the chamber. The amount of NOcontained in the effluent is one to two orders of magnitude greater thanthe concentration of NO required to achieve a vasodilatory effect intissue bath experiments using aortic strips (Morley et al. 1993).

One skilled in the art would also appreciate that the compounds of thepresent invention could be part of a complete manufactured system forportable sterilization as described in U.S. Provisional PatentApplication Nos. 60/534,395; 60/575,421; and 60/564,589, each of whichare hereby incorporated by reference in its entirety.

Use as a Pharmaceutical Agent

A number of suitable routes of administration may be employed fortreatment of animals, preferably mammals, and in particular in humans toprovide an effective dose of nitric oxide using the current invention.Oral and rectal dosage forms are preferred. However, it is possible touse subcutaneous, intramuscular, intravenous, and transdermal routes ofadministration. Of the possible solid oral dosage forms, the preferredembodiments include tablets, capsules, troches, cachets, powders,dispersions and the like. Other forms are also possible. Preferredliquid dosage forms include, but are not limited to, non-aqueoussuspensions and oil-in-water emulsions.

In one embodiment of a solid oral dosage form, a tablet includes apharmaceutical composition according to the present invention as theactive ingredient, or a pharmaceutically acceptable salt thereof, whichmay also contain pharmaceutically acceptable carriers, such as starches,sugars, and microcrystalline cellulose, diluents, granulating agents,lubricants, binders, disintegrating agents, and, optionally, othertherapeutic ingredients. Because of the acid instability of thediazeniumdiolates, it is advantageous to coat oral solid dosage formswith an enteric or delayed-release coating to avoid release of theentire dose of nitric oxide in the stomach, unless the stomach is thetherapeutic target organ.

A preferred method of coating the solid dosage form includes the use ofnon-aqueous processes to enteric or time-release coat the dosage form inorder to reduce the likelihood that nitric oxide will be released fromthe dosage form during the coating process. These non-aqueous coatingtechniques are familiar to one skilled in the art, such as thatdescribed in U.S. Pat. No. 6,576,258. A time-release coating has beendescribed in U.S. Pat. No. 5,811,121 that uses a alkaline aqueoussolution to coat solid dosage forms. This coating process would alsoserve to preserve the levels of diazeniumdiolate in the dosage form, asthe release of nitric oxide is drastically inhibited at higher pHlevels.

Rectal and additional dosage forms can also be developed by a personskilled in the art, keeping in mind the acid instability of thediazeniumdiolate class of compounds and their sensitivity to aqueoussolutions at neutral pH. One of ordinary skill in the art would be ableto develop appropriate dosage forms on the basis of knowledge withexcipients which are suitable for the desired pharmaceuticalformulation.

EXAMPLES

The following examples further illustrate the present invention. Exceptwhere noted, all reagents and solvents are obtained from AldrichChemical Company (Milwaukee, Wis.). Nitric Oxide gas is supplied byMatheson Gas Products. A detailed description of the apparatus andtechniques used to perform the reactions under an atmosphere of NO gashas been published (Hrabie et al., 1993) and is incorporated herein byreference in its entirety. The IR spectra are obtained on a Perkin Elmer1600 series FTIR. Monitoring and quantification of the evolved NO gas isperformed using a Thermo Environmental Instruments Model 42C NO—NO₂—NOxdetector calibrated daily with a certified NO gas standard. The quantityof NO released is measured in parts per billion ppb, which is determinedas follows: the NO-releasing material is placed in a chamber that has asteady stream on nitrogen gas flowing through it. The nitrogen is acarrier gas that serves to sweep any NO that is generated within thechamber into a detector. A measurement of 100 ppb means that 100molecules of NO was generated for every billion of the nitrogen gassweeping the chamber.

Example 1

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding a nitrile group. A 50 ml aliquot of DMF is dried over sodiumsulfate and then the pre-dried solvent is used to swell 2.37 g (4.42mmol Cl per g) of chloromethylated polystyrene. After 30 minutes, 3.39 g(52 mmol) KCN and 0.241 g (1.4 mmol) of KI are added. The solution isheated to 60° C. overnight. During this time the resin changes from offwhite to brick red in color. The resin is washed consecutively with 20ml portions of DMF, DMF:H₂O, H₂O, EtOH and Et₂O and allowed to air dry.The disappearance of the —CH₂—Cl stretch at 1265 cm⁻¹ and appearance ofthe nitrile absorption at 2248 cm⁻¹ is indicative of substitution.

Diazeniumdiolation: In a Parr pressure vessel, the modified resin-CN isadded to 20 ml DMF. This solution is slowly stirred and treated with 20ml (20 mmol) of 1.0 M sodium trimethylsilanolate in THF. The vessel isdegassed and charged with 54 psi NO gas. The head space is flushed withargon and the resin was washed with water, methanol and ether. Thetan/slightly orange product was allowed to air dry. This method ofdiazeniumdiolation avoids the possibility of imidate formation thatresults when using an alkoxide as the base. This material provides alarge burst of NO as shown in FIG. 1.

Example 2

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding a —OCH₃ group.

To a 50 ml solution of 1:1 DMF/MeOH, the following are added: 1.0 gchloromethylated polystyrene (4.38 mmol Cl/g), 0.014 g KI (0.08 mmol),and 1.0 ml 25% NaOMe (4.37 mmol). The solution is stirred at roomtemperature overnight. It is then vacuum filtered and washed with MeOHand ether. The product's total weight of 1.0 g is slightly higher thanthe 0.979 g theoretical weight.

Diazeniumdiolation: The resin-OCH₃ is put in a Parr pressure vessel and50 ml of 1:1 DMF/MeOH is added. While stirring, 2.0 ml 25% NaOMe (8.76mmol) is added. The solution is degassed by alternating cycles of inertgas pressurization/venting before exposure to 50 psi NO gas. Theconsumption of NO gas, an indication of the reaction of the gas with theresin, is determined the next day. In one example, it was observed that10 psi of NO gas was consumed. After vacuum filtration, washing and airdrying, the weight gain is observed. Even in the absence of weight gain,the composition produced can have a positive Greiss reaction (See,Schmidt and Kelm, 1996 for Greiss reaction) as well as NO release, asdetected by chemiluminescence.

Example 3

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding an —OC₂H₅ group. To a 50 ml solution of 1:1 DMF/EtOH, thefollowing are added: 1.0 g chloromethylated polystyrene (4.38 mmolCl/g), 0.016 g KI (0.09 mmol), and 1.7 ml 24% KOEt (4.38 mmol). Thesolution is stirred overnight at room temperature. It is then vacuumfiltered and washed with EtOH and ether. In one example, the observedweight was 1.22 g, which was slightly more than the expected 1.04 g.

Diazeniumdiolation: The resin-OC₂H₅ is placed in a Parr pressure vesselwith 50 ml solution of 1:1 DMF/MeOH, and 2.0 ml of 25% NaOMe (8.76 mmol)is added. The vessel is degassed and exposed to 60 psi NO gas overnight.The resin is then washed with methanol and ether, and air dried. In oneexample, this material had a positive Greiss reaction and spontaneouslygenerates NO under physiological conditions, as detected by an NO gasdetector, shown in FIG. 2.

Example 4

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding an —SC₂H₅ group.

In a fume hood, to 50 ml of dried DMF, the following are added: 1.00 gchloromethylated polystyrene (4.42 mmol Cl/g), 40 mg (0.24 mmol)potassium iodide and 372 mg (4.42 mmol) sodium ethanethiolate. Thismixture is stirred at room temperature for 72 hours. It is filtered andwashed with 25 ml portions of 1:1 DMF:MeOH, MeOH and Et₂O and allowed toair dry.

Diazeniumdiolation: To one gram of resin-SC₂H₅ in a Parr pressurevessel, the following are added: 25 ml of THF and 2.0 ml (8.84 mmoles)of 25% sodium methoxide. The mixture is was degassed by alternatingcharging and discharging the pressure vessel with argon before exposureto 60 psi NO gas overnight. The resin is filtered and washed with 50 mlof 0.01M NaOH, ethanol and diethyl ether. The resulting resin produces apositive Greiss reaction. When measured in a chemiluminescent NOdetector, 100 mg of resin produced 4.1×10⁻¹¹ moles NO/mg resin/min in pH7.4 buffer at room temperature over a 1 hr period.

Example 5

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding a —OSi(CH₃)₃ group. In 50 ml of dried DMF, the following areadded: 1.00 g chloromethylated polystyrene (4.42 mmol Cl/g), 10 ml of1.0 M (10 mmoles) sodium trimethylsilanolate and 100 mg (0.6 mmoles)potassium iodide. The mixture is heated to 100° C. for 24 hours.Thereafter, the resin is filtered and washed with 20 ml portions of DMF,MeOH and diethyl ether and allowed to dry in air.

Diazeniumdiolation: the following are placed in a Parr pressure vessel:1.0 g of modified resin, 30 ml DMF and 2.0 ml (8.84 mmoles) 25% sodiummethoxide. The pressure vessel is degassed and then exposed to 60 psi NOfor 24 hours. The resin is then filtered and washed consecutively withDMF, MeOH and diethyl ether. Thereafter the resin is dried in air andproduces a positive Greiss reaction. When measured in a chemiluminescentNO detector, 100 mg of resin produced 4.1×10⁻¹¹ moles NO/mg resin/min inpH 7.4 buffer at room temperature over a 40 min period.

Example 6

This example provides a method to convert commercially availablechloromethylated polystyrene into a carbon-based diazeniumdiolateincluding a diethylamine group.

A 2.17 g sample of chloromethylated polystyrene (4.42 mmol Cl⁻/g) isadded to 50 ml of DMF. To this suspension, the following are added:0.123 g (0.74 mmol) KI and 5 ml (72 mmol) diethylamine. The suspensionis stirred at 45° C. for 24 hours and then filtered and washed twicewith DMF, MeOH and ether. The resin is allowed to air dry.

Diazeniumdiolation: The following are added to a Parr pressure vessel:100 ml MeOH, 1.0 g modified resin and 2.0 ml (8.7 mmol) 25% NaOMe. Afterdegassing, the solution is exposed to 60 psi NO gas for 24 hours. Theresin is then filtered and washed with methanol and ether and allowed toair dry. Over a 150 min period, 100 mg of resin produced 9.3×10⁻¹¹ molesNO/mg resin/min in pH 7.4 buffer at room temperature.

Example 7

This example demonstrates that the NO derived from the resin originatesfrom NO donor groups attached to the resin and not to delocalized freeNO gas molecules trapped in the interstitial spaces.

A general concern working with these materials is the possibility of NObecoming trapped in the interstitial spaces within the resin, which canskew the total amount of NO produced from the resin. As a controlexperiment, 0.50 g of Merrifield resin is placed in 40 ml of a 1:1DMF/MeOH solution, degassed and exposed to 80 psi NO gas for 24 hours.The resin was then filtered, washed several times with MeOH, acetone andether. After drying in air, a 50 mg sample was placed in 5 ml of Greissreagent, which would immediately oxidize NO to nitrite and reveal thepresence of any nitrite colorimetrically. The reagent did not turn thecharacteristic purple color indicative of the presence of nitrite.Therefore, the NO that is detected from the resin is due to theformation of NO donor groups and not to trapped NO.

Example 8

This example provides a method to convert a polymer containing anaromatic ring in the backbone of the polymer e.g. poly(ethyleneterephthalate) (PET) into a carbon-based diazeniumdiolate.

In a 150 ml beaker, 2.0 g of PET pellets (Sigma-Aldrich, Milwaukee,Wis.) are treated with 10 ml of acetic acid and 10 ml of 37 wt %formaldehyde. The reaction is allowed to stir for 24 hours. Thehydroxylated PET is then filtered and washed with three 25 ml portionsof water and dried at 100° C. for one hour.

The hydroxylated PET is then suspended in 50 ml of pyridine, chilled inan ice bath, and treated with 4.67 g (2.4×10⁻² mol) of p-toluenesulfonylchloride. Two minutes after the addition of the p-toluenesulfonylchloride the reaction is allowed to warm to room temperature. Aftertwenty-four hours, the reaction is filtered and washed with two portions(25 ml) of dried DMF.

The tosylated PET is then placed in 25 ml of dried DMF and 2.03 g(3.1×10⁻² mol) of KCN is added with gentle stirring. After twenty-fourhours, the cyanomethylated PET is filtered and washed with DMF (25 ml),1:1 DMF:H₂O (25 ml), H₂O (2×25 ml), and MeOH (2×25 ml).

The cyanomethylated PET is then placed in a 300 ml Parr pressure vesselto which 25 ml of MeOH is added. The suspension is gently stirred and1.0 ml of a 1.0 M solution of sodium trimethylsilanolate intetrahydrofuran is added to the suspension. The pressure vessel ispurged and vented 10 times with argon and then charged with NO (80 psi).After twenty-four hours the diazeniumdiolated PET is filtered and washedwith 25 ml of EtOH and 25 ml of Et₂O. The release characteristics forthis compound are described in Example 14.

Example 9

In this example, a metal is coated with a siloxane and converted into anNO-releasing agent.

A piece of Nitinol, 5 mm×25 mm, is first polished with emery paper. Itis then submersed in a oxidizing solution consisting of a 1:1 mixture of1.0 M HCl and 30% H₂O₂ for 10 minutes. It is washed with water andacetone before blowing dry with argon. The clean, oxidized Nitinol stripis immersed in 6 ml of anhydrous hexadecane under an argon atmosphere.To this is added 0.2 ml dodecyltrichlorosilane, 0.2 mlchloromethylphenyltrichlorosilane and 50 μl of n-butylamine. After 24hours, the Nitinol strip is removed, dipped in ethanol to remove unboundparticles and placed in an oven at 110° C. for 15 minutes to cure. Thesiloxane modified Nitinol strip is then placed in a round bottom flaskcontaining 7 ml anhydrous hexadecane and heated to 80° C. To this isadded 0.3 ml of chlorotrimethylsilane and this is allowed to react for 1hour. The end-capped Nitinol strip is submerged in ethanol to remove anyparticles before drying at 110° C. for 20 minutes.

Next, the chloromethylphenylsiloxane Nitinol piece is placed in 15 ml ofDMF, heated to 80° C. and 10 mg of potassium cyanide, 80 mgtetrabutylammonium bromide and several catalytic grains of potassiumiodide are added. The reaction is allowed to progress overnight. TheNitinol strip is washed with ethanol before immersion in a Parr pressurevessel containing 50 ml DMF. To this is added 250 μl of sodiumtrimethylsilanolate. With gentle stirring, (avoid knocking the Nitinolstrip) the vessel is degassed and exposed to 60 psi NO gas for 24 hours.The Nitinol piece is then washed with ethanol and ether and dried underargon gas. Submersion of a piece of Nitinol treated in this manner inGreiss reagent produces a positive reaction. The Nitinol piece becomespurple in color as liberated NO is oxidized to nitrite.

Example 10

In this example, silica gel is coated with a siloxane and converted intoan NO-releasing agent. In 50 ml of toluene is placed 2.01 g of silicagel. The headspace is purged with argon. Then, 0.45 ml ofchloromethylphenyltrichlorosilane is added. The suspension is gentlystirred at room temperature overnight. The silica is then filtered andwashed with toluene and air dried. The siloxane modified silica is thenplaced in 50 ml DMF and treated with 1.0 g KI and 1.0 g KCN. Thetemperature is then raised to 110° C. for 3 hours. The silica turns andark off-red color during this phase. The silica is then filtered,washed with DMF, H₂O and methanol. It is then oven dried at 110° C. for20 minutes, and placed in a Parr pressure vessel with 50 ml THF. To thisis added 2.0 ml of 1.0 M NaOSi(CH₃)₃. The vessel is degassed and exposedto 60 psi NO gas for 24 hours. The silica is filtered, washed with THF,MeOH and Et₂O and allowed to air dry. The modified silica gel yields apositive Greiss reaction.

Example 11

In this example, the NO-releasing metal of Example 9 is treated with aprotecting group to increase the duration of NO-release. A piece ofNitinol from Example 9 is submerged in a vial containing DMF. To this isadded 50 μl of Sanger's Reagent; 2,4-dinitrofluorobenzene. The reactionis allowed to proceed at room temperature overnight. The next day theNitinol piece is removed, washed with ethanol and dried in air.

Example 12

This example demonstrates the use of carbon-based diazeniumdiolatepolymers in the ex vivo inhibition of human platelets. Blood iscollected in 0.105 M sodium citrate vacutainers from healthy volunteerswho have not consumed aspirin in the last 10 days or any NSAIDs(non-steroidal anti-inflammatory drugs) in the last 48 hours. Plateletrich plasma (PRP) is isolated by centrifuging whole citrated blood for10 min at 2000 rpm in a Sorvall clinical centrifuge. Platelet poorplasma (PPP) is prepared by centrifuging PRP for 5 minutes at 7000 rpmin a microcentrifuge. PRP is maintained in a water bath at 37° C. withgentle shaking.

Aggregometry: 5.0 ml of PRP is placed in 14 ml polypropylene tubes and20 mg/ml of the NO-releasing polymer is added. Platelets are incubatedfor 15 min at 37° C. with gentle shaking. 500 μl aliquots are placed inan aggregation cuvette and blanked against PPP in a ChronologAggregometer (37° C., 900 rpm). A baseline trace is taken for 1 min and10 μl collagen (1 mg/ml) added. Aggro-link software (Chronolog) is usedto calculate the % aggregation response after a 5 min trace. The resultsare tabulated as follows.

Group % aggregation Control 62.5 (50, 75) Thioethyl polymer  9.5 (7, 12)Nitrile polymer 15 Ethoxy polymer 42

Example 13

This example demonstrates the ability of carbon-based diazeniumdiolatepolymers to reduce the level of pathogens in stored human platelets.

PediPak platelet storage containers are filled using sterile techniquewith 3 gm of cyano-modified chloromethylated polystyrenediazeniumdiolate from Example 1, and 2 gm of ethoxy-modifiedchloromethylated polystyrene diazeniumdiolate from Example 3, (Treated)or used as is (control). Platelets from a human platelet concentrate areadded to each bag (25 ml per container) using a sterile connecting line.Each group is inoculated with 102 colony-forming units per ml (CFU/ml)of an overnight culture of S. epidermides. Aliquots from each group areimmediately removed for assessment of CFU/ml. The platelets are thenstored under the typical storage conditions of 22° C., with mildagitation. Twenty-four hours later, additional aliquots are removed forassessment of CFU/ml.

The CFU/ml is determined by serially diluting the aliquots with sterilebroth, plating the dilutions onto sterile agar and counting the numberof colonies that form on the plate after 24 hrs of incubation at 37° C.The results are tabulated as follows:

Group CFU/ml Control 5280 Treated 80

Example 14

This example shows the ability of a device comprised of a PET-derivedcarbon-based diazeniumdiolate polymer to add NO to a liquid flowingthrough the device.

An FPLC column of diameter 0.5 cm and length 10 cm is loaded with 1.2446g of the carbon based diazeniumdiolate nitrile poly(ethyleneterephthalate) from Example 8 (roughly estimated to have a surface areaof 1914 mm²/g). To ensure maximum packing the column is tapped whileinserting the polymer.

The loaded column is attached to a length of Tygon tubing and 40 ml of7.4 phosphate buffer is pumped through the column at a rate of 5 ml/min.One minute fractions are collected in 20 ml vials. Aliquots (0.5 ml) areremoved from each fraction and assayed for nitrite (assaying nitrite isan excellent surrogate for measuring NO) using the Griess assay. One mlof Griess reagent is added to the fraction in a 3 ml cuvette and theabsorbance is read at 546 nm. The results show an initial burst on NO inthe first fraction, and a decreased but stable release of NO for theremaining fractions.

μM NO released Fraction # (measured as the oxidized product) 1 101 212.5 3 7.3 4 5.4 5 6.1

Experiment 15

This example details an experiment used to study the effect of a devicesimilar to that studied in Example 14 on a mammalian organ that has beenisolated for preservation and/or transplantation, or an in vivo organundergoing an ischemic event.

To study the effects of a device designed to deliver donorless NO onorgan vascular tone, the organ is tested either in situ or isolated. Theanimals are anesthetized, heparinized, and an abdominal incision is madeto expose the kidney. Both the renal artery and vein are cannulated andthe organ is perfused with an oxygenated buffer using a peristaltic pumpat a constant flow at 85 to 95 mmHg for approximately two hours. Thesystem is monitored with flow gauges both proximal and distal to theorgan and a pressure gauge proximal to the organ. The organ is perfuseduntil the flow and pressure are stable. The flow circuit is then alteredusing two 3-way stopcocks to allow the perfusate to flow through adonorless NO releasing device. As the perfusate flows through the deviceand the effluent delivers the dissolved NO (technically NO.) to theorgan, the blood vessels dilate resulting in recordable changes in theflow (increased) and pressure readings (decreased).

To study the effects of donorless NO delivery on organ oxidative state,an ischemic-reperfusion injury is created by ligating the renal pediculewith a tourniquet for 30 to 90 minutes. During this time the renalartery proximal to the ligature is cannulated and connected to a systemthat delivers a perfusate to the organ after the appropriate time pointis reached. At the desired time-point, the tourniquet is removed, therenal vein severed, and the perfusate is pumped through the organ usinga roller pump at a constant flow at 85 to 95 mmHg for approximately twohours. The renal cortical tissue is dissected away (as the effects ofischemia/reperfusion injuries are more pronounced at the level of theproximal tubule) and homogenized. The following antioxidant enzymes andcellular components are then measured: reduced glutathione, superoxidedismutase, catalase, glutathione peroxidase. Levels of these enzymes areknown to be reduced with reperfusion injury (Dobashi et al., 2000).Protection from the oxidative damage caused by ischemia/reperfusioninsult is indicated by statistically greater levels of the antioxidativeenzyme panel above in the NO-treated group versus those levels in thecontrol kidneys not receiving NO.

The foregoing disclosure of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Many variations andmodifications of the embodiments described herein will be apparent toone of ordinary skill in the art in light of the above disclosure. Thescope of the invention is to be defined only by the claims appendedhereto, and by their equivalents.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention.

Example 16

Preparation of a Contact Lens Case Made of Pet, Modified as Described inthe instant specification and analysis of the its antimicrobialproperties.

A standard contact lens case is manufactured using PET using the mostappropriate method as known by one skilled in the art. The case istreated with acetic acid and 37% wt formaldehyde, as described inExample 8. The case is suspended in pyridine, chilled in an ice bath,and treated with at least 4.67 g of p-toluenesulfonyl chloride. Twominutes after the addition of the p-toluenesulfonyl chloride thereaction is allowed to warm to room temperature. After twenty-fourhours, the contact lens case is removed and washed with two portions ofdried DMF.

The tosylated PET is then placed in an appropriate volume of dried DMFand least 2.03 g (3.1×10⁻² mol) of KCN is added with gentle stirring.After twenty-four hours, the cyanomethylated PET is filtered and washedwith DMF, 1:1 DMF:H₂O, H₂O, and MeOH.

The cyanomethylated PET is then placed in a 300 ml Parr pressure vesselto which an appropriate volume of MeOH is added. The suspension isgently stirred and at least 1.0 ml of a 1.0 M solution of sodiumtrimethylsilanolate in tetrahydrofuran is added to the suspension. Thepressure vessel is purged and vented 10 times with argon and thencharged with NO (80 psi). After twenty-four hours the diazeniumdiolatedPET contact lens case is removed and washed with sufficient amounts ofEtOH and Et₂O.

Several diazeniumdiolated contact lens cases, and an equal number ofcontrol cases are were gassed with 80 psi nitrogen, instead of NO, andthen challenged with a strain or strains of bacteria commonly found tocontaminate contact lens cases including but not limited to P.aeruginosa, S. aureus, S. epidermidis, Bacillus spp., Propionibacteriumspp., Corynebacterium spp., and Mycobacterium spp. After a 24 hourincubation period, the lens cases are rinsed gently three times in amild buffer, and quantitatively assessed for the degree of bacterialcolonization, such assessment including but not limited to scanningelectron microscopy, removal of adhered bacteria by physical(sonication) or chemical (detergent removal) means, and/or countingmicroorganisms by microscopy or spectophotometry, as known to those ofskill in the art. The antimicrobial effect of the diazeniumdiolatedcontact lens cases is indicated by a statistically significant decreasein the amount of adhered bacteria versus the amount found on the controlcontact lens cases.

Experiment 17

Analysis of the resistance of NO-releasing surfaces to the formation ofviable microbial biofilms. Glass disks (5 mm) are coated with asiloxane-based NO-releasing polymer of the present invention. Controldisks are coated but gassed with nitrogen instead of NO. Thediazeniumdiolated and control disks are placed in the wells of 96 wellmicrotiter plate where bacterial biofilms are then grown as describedpreviously (Yarwood et al. 2004; Hasset et al. 1999; Pitts et al. 2003).Overnight cultures of bacteria in Tryptic Soy Broth (TSB) (or speciesspecific medium) are diluted 1:10 in sterile TSB. The 96 well plates arecoated with Fetal Calf Serum, and washed twice with PBS, in order tocreate a conditioning film. Subsequently, wells are filled with 180 μlsterile TSB. Wells are then inoculated with 20 μl of the suspension ofthe bacteria under study. The plates are incubated at 37° C. for 24hours. Planktonic cells and medium are removed by aspiration after whichthe wells are washed twice with sterile PBS.

Viability of the cells comprising the biofilm is assayed as describedpreviously (Pitts et al. 2003) with minor adjustments. 100 μl prewarmedPBS containing 1% glucose, 0.5 μg/ml13-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)(5 mg/ml stock in PBS, store in aliquots at −20° C.), 1 μM menadione(from 1 mM stock in acetone) is added to each well. The plates areincubated statically at 37° C. for 1 hour, after which the wells areaspirated. Then, 100 μl acid isopropanol (5% 1N HCl) is added todissolve the formazan crystals. The plates are incubated at ambienttemperature for 10 min while shaking. The solution is then transferredto clean 96 well plates and the absorbance at 550 nm is determinedutilizing a spectrophotometric plate reader to measure the number ofmetabolically active bacteria in the biofilm.

A statistically significant decrease in the absorbance readings at 550nm in the wells containing NO-releasing disk coatings versus the controldisk coatings indicates a reduction in viability of the biofilm. Similarexperiments are performed with fungal biofilms and mixedbacterial/fungal biofilms.

Example 18

The resistance of NO-releasing surfaces to platelet adhesion. Glasscoverslips are coated using the same procedure as described in Example10. Control coverslips are gassed with nitrogen instead of NO. Controland NO-releasing slides are sealed into a flow cell mounted on the stageof a fluorescent microscope with a video recording camera and wholehuman blood is circulated through the cell at 37° C. under high shearconditions (1000 s-1), and fluorescence is monitored. Deposition ofplatelets to the surface is indicated by white fluorescent spots on thevideo image. Experiments are controlled such that the same blood donoris tested using NO-releasing and control coatings. An effectiveantiplatelet coating is indicated by zero fluorescence with less than 5%area coverage for the NO-releasing coating versus a strong fluorescentimage, with greater than 20% area coverage for the control coatings.

Example 19

Demonstration of the ability of NO-releasing coatings to enhance thegrowth of endothelial cells on artificial surfaces. Glass slides arecoated with a nitrile-modified (see Example 1) siloxane diazeniumdiolatemonolayer polymer (similar to Example 9), or the identical polymer thatis gassed with nitrogen instead of NO (as control that does not releaseNO). Slides are sterilized in alkalinized 70% ethanol for at least 20min. The slides are placed in respective sterile Petri dishes. C166bovine endothelial cells are seeded in the Petri dishes at 1×10⁴cells/ml, using 4 malls. The samples are incubated at 37° C. under 5%CO₂. After 24 hours, the number of endothelial cells adhering to thecoated slide is counted by the following method. The slides aretransferred to fresh Petri dishes where the cells are released from theslide using EDTA and trypsinization extraction, followed by washing,staining, centrifugation to concentrate the cells, and counting using ahemocytometer. These experiments demonstrate the ability of anNO-releasing coating to accelerate the endothelialization of a foreignsurface.

Coating Group Cells per ml of extract Control 2.7 × 10⁵ NO-releasing 1.3× 10⁶

Example 20

Evaluation of a cardiovascular stent coated with an NO-releasing coatingas described in the instant application. A stent is coated as describedin the present invention. The stents are implanted using the porcinecoronary artery restenosis model according to the guidelines andprocedures of Schwartz and Edelman, 2002. Three experimental groupsincluding an NO-releasing coated stent, a non-NO-releasing coated stent(i.e. coated but not exposed to NO gas according to the presentinvention), and a plain metal stent, are implanted into animals treatedwith antiplatelet medication (aspirin and clopidogrel, 24 hrs presurgery and continuing). Stents with a stent:artery ratio of 1.0 to 1.1are used. The implantation of the stents is performed under anesthesia.Stented arteries, approximately 10 per experimental group, are evaluatedat 7 days, 28 days, and 3 months.

Efficacy of the NO-releasing coating is determined by the absence ofthrombi and a statistically significant reduction of neointimal growthcompared to bare stents, using the quantitative and semi-quantitativemethods described in Schwartz and Edelman.

The invention claimed is:
 1. A composition comprising a carbon-baseddiazeniumdiolate compound attached to at least one phenyl-containingpolymer, and having the formula:R³—C(R¹)_(x)(N₂O₂R²)_(y) wherein x is an integer from 0 to 2 and y is aninteger from 1 to 3, and the sum of x plus y equals 3; wherein R¹ is notan imidate or thioimidate; wherein R² is selected from the groupconsisting of a countercation and a protecting group on the terminaloxygen; and wherein R³ is a phenyl of said phenyl-containing polymer. 2.A composition comprising a carbon-based diazeniumdiolate compoundattached to at least one phenyl-containing polymer that comprises apolymer backbone, wherein the phenyl of said phenyl-containing polymeris pendant from the backbone of said polymer, and wherein saidcomposition has the following general formula:

wherein R¹ not an imidate or thioimidate; and wherein R² is selectedfrom the group consisting of a countercation and a protecting group onthe terminal oxygen.
 3. A medical device coating comprising a nitricoxide-releasing polymer, wherein said nitric oxide-releasing polymercomprises the composition of claim
 1. 4. The medical device coating ofclaim 3, wherein said medical device is selected from the groupconsisting of a vascular stent, a vascular graft, a catheter, a wound abandage, a blood collection bag, a blood component storage bag, anextracorporeal membrane oxygenation (ECMO) circuit, an internalmonitoring device, an external monitoring device, and a device thatcomes in contact with mammalian tissue in vivo, in vitro, or ex vivo. 5.A medical device, wherein all or part of the device comprises a nitricoxide-releasing polymer that comprises the composition of claim
 1. 6.The composition of claim 1, wherein the at least one phenyl-containingpolymer comprises one or more polystyrene units cross-linked withdivinylbenzene.
 7. The composition of claim 2, wherein the at least onephenyl-containing polymer comprises one or more polystyrene unitscross-linked with divinylbenzene.
 8. A medical device coating comprisinga nitric oxide-releasing polymer, wherein said nitric oxide-releasingpolymer comprises the composition of claim
 2. 9. The medical devicecoating of claim 8, wherein said medical device is selected from thegroup consisting of a vascular stent, a vascular graft, a catheter, awound dressing, a bandage, a blood collection bag, a blood componentstorage bag, an extracorporeal membrane oxygenation (ECMO) circuit, aninternal monitoring device, an external monitoring device, and a devicethat comes in contact with mammalian tissue in vivo, in vitro, or exvivo.
 10. A medical device, wherein all or part of the device comprisesa nitric oxide-releasing polymer that comprises the composition of claim2.
 11. The composition of claim 6, wherein R¹ is selected from the groupconsisting of a cyano group; an ether group, a tertiary amine, and athioether.
 12. The composition of claim 7, wherein R¹ is selected fromthe group consisting of a cyano group; an ether group, a tertiary amine,and a thioether.
 13. The composition of claim 6, wherein R¹ is a cyanogroup.
 14. The composition of claim 7, wherein R¹ is a cyano group. 15.The composition of claim 6, wherein the carbon-based diazeniumdiolatecompound attached to at least one phenyl-containing polymer comprisesthe following moiety:

wherein R² is a countercation.
 16. The composition of claim 15, whereinR² is Na.
 17. A medical device coating comprising a nitricoxide-releasing polymer, wherein said nitric oxide-releasing polymer isthe composition of claim
 15. 18. The medical device coating of claim 17,wherein said medical device is selected from the group consisting of avascular stent, a vascular graft, a catheter, a wound dressing, abandage, a blood collection bag, a blood component storage bag, anextracorporeal membrane oxygenation (ECMO) circuit, an internalmonitoring device, an external monitoring device, and a device thatcomes in contact with mammalian tissue in vivo, in vitro, or ex vivo.19. A medical device, wherein all or part of the device comprises anitric oxide-releasing polymer that comprises the composition of claim15.
 20. A system for localized release of nitric oxide to a target site,the system comprising: the composition of claim 6; whereby decompositionof a carbon-bound diazeniumdiolate moiety to produce nitric oxide occursunder physiological conditions and does not produce a nitrosamine donormolecule.